Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, to actually control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
This technology has progressed extremely rapidly in recent years, and a variety of exogenous proteins has been expressed in a variety of hosts. By way of example, some of the eukaryotic proteins so-produced include: proinsulin (Naber, S. et al., Gene 21: 95-104 [1983]); interferons (Simon, L. et al., Proc. Nat. Acad. Sci. U.S.A., 80: 2059-2062 [1983] and Derynck, R. et al., Nucl. Acids Res. 1: 1819-1837 [1983]); and growth hormone (Goeddel, D., et al., Nature 281: 544-548 [1979]). (These publications and other referenced materials have been included to provide additional details on the background of the pertinent art and, in particular instances, the practice of invention, and are all incorporated herein by reference.)
For some time, it has been clear that the mammalian immune response was due primarily to a series of complex cellular interactions, coined the "immune network". While it remains clear that much of the response does in-fact revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins (e.g., the so-called lymphokines) play a critical role in controlling these cellular interactions.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the basic differentiation of pluripotential hematopoietic stem cells into the vast number of progenitors of the diverse cellular lineages responsible for the immune response. Cell lineages important in this response include two classes of lymphocytes: B cells that can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T cells of various subsets that induce or suppress B cells and some of the other cells (including other T cells) making up the the immune network.
Another important cell lineage is the mast cell--a granule-containing connective tissue cell located proximate to capillaries throughout the body, with especially high concentrations in the lungs, skin, gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy related disorders, particularly anaphylaxis, as follows. Briefly stated, once certain antigens crosslink special immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases the mediators (e.g., histamine, serotonin, heparin, kinins, etc.) which cause anaphylactic and some other allergic reactions.
Research to better understand (and thus potentially treat therapeutically) allergy, anaphylaxis and other immune disorders, through the study of mast cells, T cells and the other cells involved in the immune response, has been hampered by the general inability to maintain these cells in vitro. However, several immunologists recently discovered that such cells could be isolated and cultured by growing them on secretions from other cells, e.g., conditioned media from Concanavalin A (Con A) stimulated splenic lymphocytes. It has now become clear from this work that the generation of cell clones is dependent on specific factors, such as lymphokines.
All blood cell types are continuously generated in the adult vertabrate bone marrow through the growth and differentiation of the hierarchy of hematopoietic progenitor cells. At the apex of this hierarchy is the pluripotent stem cell which can repopulate a lethally irradiated animal for most, if not all, immunological cell types (e.g., red cells, platelets, lymphocytes, various granulocytes and monocytes/macrophages). The pluripotent cell not only has the capacity to regenerate the pluripotent stem cell compartments (self-renewal), but also gives rise to progenitor cells committed to development along one particular lineage pathway. All the progeny of a particular committed stem cell share the same lineage commitment as the parent cell (Metcalf, D., "Hemopoietic Colonies", Springer Publishing Co., New York, N.Y., [1977]).
In vito studies on hematopoiesis have shown that a number of soluble cloning stimulating factors (CSF) can regulate the growth of these various progenitor cells. Some of these factors have been partially purified and shown to specifically effect stem cells belonging to a particular cell lineage. For example, erthropoietin stimulates more differentiated members of the erythroid hierarchy (Miyake, T., et.al., J. Biol. Chem. 252: 5558, [1977]), whereas another factor (colony stimulating factor-macrophage or CSF-1) preferentially stimulates macrophage growth in semi-solid cultures of bone marrow cells (Stanley, E., and Heard, P., J. Biol. Chem. 252: 4305, [1977]). Another type of growth factor stimulates hematopoietic colonies consisting of single cell types and mixtures of erythrocytes, megakaryocytes, granulocytes, mast cells and monocyte/macrophages (Iscove, N. et.al., J. Cell. Physiol. Suppl., 1: 65-78, [1982]). The range of progenitor cells responsive to this second type of factor indicates that it may be a multi-lineage cellular growth factor (multi-CSF) effecting various committed progenitor cells, and perhaps pluripotential stem cells as well.
Other factors include, for example, interleukin-1 (IL-1), a factor released from macrophages, which induces replication of thymocytes and peripheral T cells (Mizel, S. et al., J. Immunol. 120: 1497-1503 [1978]). Similarly, interleukin-2 (IL-2) and interleukin-3 (IL-3), are both of wherein released by certain stimulated lymphocytes. A very significant characteristic of IL-2 is the ability to support the continuous growth of certain T cells in vitro (Farrar, et al., Ann. N.Y. Acad. Sci. 332: 303-15 [1979]). Likewise, an important characteristic of IL-3 is the ability to support the growth of cell lines having the phenotypic characteristics of mast cells (Ihle, J., et al., Immunological Rev. 63: 5-32 [1982]). A number of other cellular growth properties have been ascribed to IL-3 as well (see, Ihle, J. et. al., J. Immunol. 131: 282-287 and 129: 2431 [1983]).
While both mouse IL-2 and IL-3 have been at least partially characterized biochemically (Gillis, S. et al., J. Immunol. 124: 1954-1962 [1980] and Ihle, J. et al., J. of Immunol. 129: 2431-2436 [1982], respectively), Il-2 is presently the accepted primary factor responsible for T-cell growth, whereas the protein(s) responsible for mast cell growth factor (MCGF) and CSF activity have not been agreed upon to the same extent. It is now though that mouse IL-2 has a molecular weight (probably as a dimer) of approximately 30-35,000 (Simon, P. et al., J. Immunol. 122: 127-132 [1979]--although some variations are recognized (Robb, R. and Smith, K., Molec. Immun. 18: 1087-1094 [1981]); and human IL-2 apparently has a molecular weight of about 15,000 (Gillis, S. et al., Immun. Rev. 63: 167-209 [1982]. Moreover, a cDNA clone coding for human IL-2 has recently been reported (Taniguchi, T., et al., Nature 302: 305-310 [1983]). On the other hand, mouse mast cell growth factors are presently reported as having molecular weights of 45,000 (Nabel, et al., Nature, 291: 332-334 [1981], of 35,000 (Yung, Y., et al., J. Immunol. 127: 794-799 [1981]and of 28,000 (Ihle, J., et al., J. of Immunol. 129: 1377-1383 [1982]). Similar discrepancies surround the CSFs'.
Although the molecular weight differences could be perhaps partially explained by varying amounts of glycosylation, clarification of the issue requires additional structural data, e.g., substantially full length sequence analysis of the molecules in question. Protein sequencing offers, of course, a possible means to solve the problem, but it is very difficult work experimentally and often cannot provide completely accurate, nor full-length, amino acid sequences. Moreover, having the capability of making bulk quantities of a polypeptide exhibiting mammalian MCGF or CSF activity will greatly facilitate the study of the biology of mast cells and other cells involved in the immune response; e.g., by minimizing the necessity of relying on Con A conditioned media for stimulating cell growth. Accurate and complete sequence data on a mouse MCGF and CSF will also serve to simplify the search for human MCGF and CSF proteins. Finally, additional information on any lymphokine will help in evaluating the roles of the various growth factors and cells of the immune network, providing insight into the entire immune system--with the concomitant therapeutic benefits.
Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding for, and amino acid sequences of, proteins exhibiting MCGF or CSF activity, as well as a simple and economic method of making substantial quantities of such materials. The present invention fulfills these needs.